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Publications and source records attributed to H I Mosberg.
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In an effort to explore structural features affecting receptor recognition in a series of conformationally restricted tetrapeptides related to the cyclic, delta opioid receptor-selective analogue, [formula: see text] electronic, lipophilic, and steric effects at the Phe3 residue were assessed by substitution at different positions of the side-chain aromatic ring by halogens, alkyl, hydroxyl, and nitro groups. Effects on opioid receptor binding affinity and selectivity were determined. The results, which are generally consistent with reports of analogous modifications in linear and cyclic pentapeptide enkephalins, indicate that steric, lipophilic, and electronic properties are all important determinants of delta opioid receptor recognition. Specifically, modifications which increase lipophilicity or exert electron-withdrawing effects on the aromatic ring enhance binding affinity, while hydrophilic, bulky, or electron-releasing modifications are detrimental. These observations are in excellent agreement with quantitative structure-activity relationship (QSAR) results reported for Phe4 modifications in linear opioid pentapeptide enkephalin analogues, suggesting that the Phe3 tetrapeptide side chain and the Phe4 pentapeptide side chain interact with the same delta receptor binding subsite.
The cyclic peptide [2,6-dimethyl-Tyr1,D-Pen2,D-Pen5]enkephalin (2) was synthesized by solid-phase techniques and contains the optically pure unnatural amino acid 2,6-dimethyltyrosine (DMT) as a replacement for the Tyr1 residue of [D-Pen2,D-Pen5]enkephalin (DPDPE, 1). This structural modification resulted in a 10-fold increase in the potency of 2 at the delta opioid receptor and a 35-fold increase in potency at the mu receptor while substantial delta receptor selectivity was maintained. In addition, 2 was 86-fold more effective than 1 at inhibiting electrically stimulated contractions of the mouse vas deferens. In the hot plate test, 2 was 7-fold more potent than 1 after intracerebroventricular administration in the mouse. While 1 was inactive following systemic administration of doses as high as 30 mg/kg, subcutaneous administration of 2 significantly inhibited writhing with an ED50 of 2.6 mg/kg. These results demonstrate that the potency and systemic activity of DPDPE are significantly increased by replacement of Tyr1 with DMT.
The feasibility of targeting prolidase as a peptide prodrug-converting enzyme has been examined. The enzymatic hydrolysis by prolidase of substrates for the peptide transporter L-alpha-methyldopa-pro and several dipeptide analogues without an N-terminal alpha-amino group (phenylpropionylproline, phenylacetylproline, N-benzoylproline, and N-acetylproline) was investigated. The Michaelis-Menten parameters Km and Vmax for L-alpha-methyldopa-pro are 0.09 +/- 0.02 mM and 3.98 +/- 0.25 mumol/min/mg protein, respectively. However, no hydrolysis of the dipeptide analogues without an N-terminal alpha-amino group is observed, suggesting that an N-terminal alpha-amino group is required for prolidase activity. These results demonstrate that prolidase may serve as a prodrug-converting enzyme for the dipeptide-type prodrugs, utilizing the peptide carrier for transport of prodrugs into the mucosal cells and prolidase, a cytosolic enzyme, to release the drug. However, a free alpha-amino group appears to be necessary for prolidase hydrolysis.
The previously described cyclic delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH (JOM-13) was modified at residue 3 by incorporation of both natural and unnatural amino acids with varying steric, electronic, and lipophilic properties. Effects on mu and delta opioid receptor binding affinities were evaluated by testing the compounds for displacement of radiolabeled receptor-selective ligands in a guinea pig brain receptor binding assay. Results obtained with the bulky aromatic 1-Nal3 and 2-Nal3 substitutions suggest that the shape of the receptor subsite with which the side chain of the internal aromatic residue interacts differs for delta and mu receptors. This subsite of either receptor can accommodate the transverse steric bulk of the 1-Nal3 side chain but only the delta receptor can readily accept the more elongated 2-Nal3 side chain. Several analogs with pi-excessive heteroaromatic side chains in residue 3 were examined. In general, these analogs display diminished binding to mu and delta receptors, consistent with previous findings for analogs with residue 3 substitutions of modified electronic character. Several analogs with alkyl side chains in residue 3 were also examined. While delta receptor binding affinity is severely diminished with Val3, Ile3, and Leu3 substitutions, Cha3 substitution is very well tolerated, indicating that, contrary to the widely held belief, an aromatic side chain in this portion of the ligand is not required for delta receptor binding. Where possible, comparison of results in this delta-selective tetrapeptide series with those reported for analogous modification in the cyclic delta-selective pentapeptide [D-Pen2, D-Pen5]enkephalin (DPDPE) and linear pentapeptide enkephalins reveals similar trends.
Previous studies from our laboratory have indicated that i.c.v. pretreatment of mice with the novel, selective opioid delta receptor antagonists, [D-Ala2,Leu5,Cys6]enkephalin (DALCE) and naltrindole-5'-isothiocyanate (5'-NTII), differentially antagonized the direct antinociceptive effects of [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2]deltorphin II (DELT). These findings, and others, suggested the existence of subtypes of opioid delta receptors which could be classified as activated by DPDPE and DALCE sensitive (delta 1 receptor), or selectively activated by DELT and 5'-NTII sensitive (delta 2 receptor). The present study has extended these observations to the characterization of delta-mediated antinociception effects of DPDPE and DELT after i.t. administration in mice using pretreatment with DALCE and 5'-NTII in order to selectively antagonize the delta subtypes. Additionally, the acute antinociceptive actions of DALCE itself were studied to ensure activity of this compound at the spinal level. The respective antinociceptive A50 value (95% CL) for i.t. DPDPE, DELT and DALCE were 19.0 (12.9-28.1), 19.3 (16.1-23.1) and 2.0 (1.4-3.0) nmol. The delta antagonist, N,N-diallyl-Try-Aib-Aib-Phe-Leu-OH (ICI 174,864) (where Aib is alpha-aminoisobutyric acid) blocked the antinociceptive effects of DPDPE and DELT, but not those of i.t. morphine or [D-Ala2,NMPhe4,Gly-ol5]enkephalin (DAMGO), indicating that the observed antinociceptive effects of DPDPE and DELT were delta mediated. Pretreatment 24 hr before testing with graded doses of i.t. 5'-NTII blocked the i.t. antinociceptive effects of DPDPE and DELT, although at least a 10-fold higher dose of 5'-NTII was needed to produce equivalent antagonism of DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS)
The present study has characterized the antinociceptive response to cold water swim-stress (CWSS) in mice using opioid-selective antagonists as well as tolerance and cross-tolerance approaches. Mice subjected to CWSS using water at 5 degrees C for 3 min showed a marked antinociceptive response in the tail-flick test, which reached approximately 90% after +10 min, and which persisted for 15 to 20 min. This antinociceptive response (at +10 min) was antagonized by naloxone or by the delta antagonist ICI 174,864. Additionally, the CWSS response was antagonized by the opioid delta 2 antagonist, naltrindole-5'-isothiocyanate, but not by the delta 1 antagonist, [D-Ala2,Leu5,Cys6]enkephalin, or by the mu antagonist, beta-funaltrexamine or by the kappa antagonist, norbinaltorphimine. Although the CWSS-induced antinociceptive effect was blocked by some delta antagonists and tolerance resulted from the CWSS-induced response, the decrease in body temperature after each CWSS exposure was not affected by the opioid antagonists and reliably occurred in CWSS-tolerant mice, suggesting that the observed antinociception was independent of changes in body temperature. In mice rendered tolerant to the antinociceptive actions of the mu agonist, [D-Ala2,NMPhe4,Gly-ol] enkephalin, or to [D-Pen2,D-Pen5]enkephalin (predominantly a delta 1 agonist), the CWSS-induced antinociceptive response was unaltered. In contrast, in mice tolerant to the delta 2 agonist, [D-Ala2,Glu4]deltorphin, the CWSS-induced antinociceptive response was markedly and significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)
Recently, subtypes of the opioid delta receptor have been identified. It is not known, however, if a subtype of opioid delta receptor can be associated with the known modulatory action of delta agonists on mu-mediated antinociception. Thus, the present study has used the delta subtype-selective antagonists, [D-Ala2,Leu5,Cys6]enkephalin (DALCE) (delta 1 antagonist) and naltrindole-5'-isothiocyanate (5'-NTII) (delta 2 antagonist) in an effort to determine whether the positive and negative modulation of morphine antinociception produced by opioid delta agonists was the result of activity at specific subtypes of opioid delta receptors. Intracerebroventricular morphine produced a dose-related antinociceptive effect which was not antagonized by coadministration of the delta antagonist, ICI 174,864, or by pretreatment 24 hr before testing with the DALCE or 5'-NTII. Coadministration with morphine of a nonantinociceptive dose of DPDPE or [D-Ala2,Glu4]deltorphin resulted in a leftward displacement of the morphine dose-effect curve (i.e., positive modulation), whereas coadministration of a nonantinociceptive dose of [Met5]enkephalin with morphine resulted in a rightward displacement of the morphine dose-effect curve (i.e., negative modulation). Both the positive and the negative modulatory actions were antagonized when the experiment was conducted in the presence of the delta antagonist, ICI 174,864, or when the mice were pretreated with the delta 2 antagonist, 5'-NTII. In contrast, pretreatment with the delta 1 antagonist, DALCE, failed to affect either the positive or the negative modulatory actions of these delta agonists on morphine antinociception. The data suggest the involvement of an opioid delta 2 receptor in the modulation of morphine antinociception.
Dissociation constants (KB) for naloxone inhibition of the actions of DPDPE in the mouse isolated vas deferens preparation (inhibition of electrically induced twitch) were determined at five temperatures ranging between 25 and 40 degrees C. The values of KB tended to increase with temperature over the range examined, indicating that the affinity of naloxone for the opioid delta receptor is an inverse function of temperature. Using these data, the thermodynamic quantities delta G zero' (change in free energy), delta H zero' (change in enthalpy) and delta S zero' (change in entropy) were calculated from a van't Hoff plot of in (KB) against 1/T. The thermodynamic quantities determined in this study in vivo (delta G zero' = -10.59 kcal mol-1, delta H zero' = -15.73 kcal mol-1 and delta S zero' = -0.0168 kcal mol-1 zero K-1) are consistent with data reported from radioligand binding studies in vitro and suggest that the interaction between naloxone and the opioid delta receptor in the mouse isolated vas deferens is enthalpy driven. These data represent the first evaluation of the thermodynamics of opioid antagonist/receptor interaction in a physiological assay.
Possible involvement of adenosine, as a secondary neurotransmitter, in opioid modulation of nociception and gastrointestinal function was investigated in mice. Inhibitory actions of theophylline, a nonselective adenosine receptor antagonist, were evaluated against effects evoked by opioid receptor-selective agonists administered at spinal or supraspinal sites. Intrathecal administration of theophylline significantly inhibited antinociceptive actions produced by intrathecal (i.th.) injections of morphine, [D-Ala2, NMPhe4, Gly-ol] enkephalin (DAMGO), [D-Pen2, D-Pen5] enkephalin (DPDPE) and beta-endorphin as measured with the warm water tail-flick assay. The rank order of rightward displacement of i.th. agonist dose-response curves by theophylline (i.th.) was DPDPE (greatest) > DAMGO > morphine > beta-endorphin. Theophylline was less effective as an inhibitor in the hot-plate assay. Additionally, i.th. administration of theophylline inhibited antinociceptive effects evoked by i.c.v. administration of opioids. The rank order of rightward displacement of dose-response curves after i.c.v. opioid administration was DAMGO (greatest) > beta-endorphin > morphine > DPDPE. In contrast to the effectiveness of theophylline administered i.th., theophylline coadministered i.c.v. with opioid agonists did not inhibit opioid-induced antinociception. Neither i.th. nor i.c.v. theophylline altered inhibitory effects on gastric emptying and gastrointestinal propulsion produced by i.th. or i.c.v. administration of selective opioid agonists. These data provide additional support for involvement of spinal adenosine as a secondary neurotransmitter in opioid antinociceptive processes associated with local spinal reflexes as well as in descending antinociceptive processes. Adenosine was not involved in modulation of opioid-activated gastrointestinal outflow pathways at either spinal or supraspinal levels.
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Substantial evidence has been accumulated which suggests that opioid delta receptors may be distinguished on the basis of their involvement in the modulation (i.e., increase or decrease in potency) of mu-mediated antinociception. On this basis, it has been hypothesized that some opioid delta receptors exist within a functional complex with mu receptors (delta complexed (delta cx) receptors) while other delta sites do not (delta non-complexed (delta ncx) receptors). Recent work with [D-Ala2,Leu5,Cys6]enkephalin (DALCE) has demonstrated that this compound produces initial antinociceptive actions, does not modulate morphine antinociception and appears to bind irreversibly to the delta ncx site, presumably by means of thiol-disulfide exchange between the receptor and the cysteine sulfhydryl group. To determine if a structural basis exists for actions at the hypothesized delta ncx receptor, in the present study we report the synthesis and pharmacological characterization of [D-Ala2,Leu5,Ser6] enkephalin (DALES), a close structural analogue of DALCE. If a structural basis for action at the delta ncx site exists, then DALES would be predicted to produce antinociception, fail to modulate morphine antinociception and, since it lacks the free sulfhydryl group present in DALCE, fail to exhibit irreversible antagonistic actions; these predictions were supported. Additionally, pretreatment with DALCE at -24 h, but not with DALES, blocked DALES-induced antinociception. These observations in vivo support the concept of a structural basis for activity at the hypothesized delta ncx site and suggest that DALES, like DALCE, may be a useful probe for pharmacological characterization of putative delta receptor subtypes.
The requirement for a free alpha-amino group for the intestinal peptide carrier-mediated transport was investigated. A series of dipeptide analogues without the N-terminal alpha-amino group [including phenylpropionylproline, phenylacetylproline, N-benzoylproline, phenylacetyl-alpha-methyldopa, and hippuric acid (N-benzoylglycine)] were studied in the perfused rat intestinal segment. The absorption of phenylpropionylproline, phenylacetyl-alpha-methyldopa, and N-benzoylproline was concentration dependent. The transport parameters (mean +/- SD) of phenylpropionylproline and N-benzoylproline were as follows: Jmax*, 0.037 (+/- 0.019) mM; Km, 0.045 (+/- 0.027) mM; Pc*, 0.830 (+/- 0.130); and Pm*, 0.673 +/- 0.049; and Jmax*, 1.34 (+/- 0.24) mM; Km, 1.31 (+/- 0.30) mM; Pc*, 1.02 (+/- 0.11); and Pm*, 0; respectively. The intestinal permeabilities of phenylpropionylproline, phenylacetylproline, N-benzoylproline, and hippuric acid (N-benzoylglycine) were significantly reduced by dipeptides and cephradine. These results strongly suggest that these dipeptide analogues, without an alpha-amino group, are transported by the peptide carrier and provide more direct evidence that a free alpha-amino group is not absolutely essential for the mucosal-cell peptide carrier-mediated transport.
The previously described cyclic, delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH, where Pen, penicillamine, is beta-beta-dimethylcysteine, was modified at residues 2 and 4 by varying combinations of D- and L-Cys and D- and L-Pen, and effects on mu and delta opioid receptor binding affinities and on potency in the mouse vas deferens (MVD) smooth muscle assay were evaluated. A comparison was drawn between consequences of alterations in this series of analogs and those of analogous modifications in the related cyclic pentapeptide series which includes the highly delta receptor-selective [D-Pen2,D-Pen5]enkephalin, DPDPE. Unlike effects observed in the cyclic pentapeptide series, the mu receptor binding affinities of the cyclic tetrapeptides are not dramatically influenced by substitution of Pen for Cys at residue 2. Conversely, while binding of the pentapeptides is only slightly affected by alteration of the chirality of the carboxy-terminal residue, modification of stereochemistry at the carboxy terminus in the tetrapeptides critically alters binding behavior at both mu and delta sites. In contrast with the pentapeptide series, the tetrapeptides appear to be highly dependent upon primary sequence for binding and activity, as only the lead compound binds with high affinity to the delta site. Results suggest that the less flexible cyclic tetrapeptides, lacking the Gly3 residue, display more stringent structural requirements for binding and activity than do the corresponding cyclic pentapeptides.
The present study has investigated the direct opioid delta receptor-mediated antinociception produced by i.c.v. administration of the highly selective delta agonists, [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2]deltorphin II, as well as that of the less delta-selective [D-Ser2,Leu5,Thr6]enkephalin (DSLET), by using two novel nonequilibrium opioid antagonists, [D-Ala2,Leu5,Cys6] enkephalin (DALCE) and naltrindole 5'-isothiocyanate (5'-NTII). At times ranging from 8 to 48 hr after a single i.c.v. pretreatment of mice with 5'-NTII, the antinociceptive effects of [D-Ala2] deltorphin II were significantly antagonized. In contrast, 5'-NTII pretreatment at times between 10 min and 24 hr failed to antagonize the antinociceptive effects of DPDPE. Previous studies have shown that pretreatment with i.c.v. DALCE produces a dose- and time-related antagonism of DPDPE, but not morphine, antinociception. However, pretreatment with i.c.v. DALCE failed to antagonize the antinociceptive effects of [D-Ala2]deltorphin II. Similarly, i.c.v. administration of DSLET produced time- and dose-related antinociception which was partially antagonized by either beta-funaltrexamine (beta-FNA) or by ICI 174,864 (N,N-dialyl-Tyr-Aib-Aib-Phe-Leu-OH), suggesting mixed activity at mu and delta receptors. ICI 174,864 produced essentially complete antagonism of DSLET antinociception in beta-FNA-pretreated mice. Pretreatment with 5'-NTII (at -8 to -48 hr), blocked the antinociception produced by DSLET in control or in beta-FNA-pretreated mice. In contrast, pretreatment with DALCE failed to antagonize the antinociception produced by i.c.v. DSLET in either control or in beta-FNA-pretreated mice.(ABSTRACT TRUNCATED AT 250 WORDS)
This study has investigated the development of antinociceptive tolerance to, and cross-tolerance between, two highly selective delta agonists, [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2] deltorphin II as well as to [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO), a highly selective mu agonist, in mice. Intracerebroventricular administration of DPDPE, [D-Ala2]deltorphin II and DAMGO each produced an antinociceptive effect. Pretreatment with i.c.v. DPDPE twice daily for 3 days resulted in tolerance to DPDPE as shown by a 4.8-fold rightward shift in the dose-response curve. In contrast, in DPDPE pretreated mice, the dose-response lines for [D-Ala2]deltorphin II and DAMGO were not altered when compared to those obtained in naive animals. The development of tolerance was also shown by pretreating mice with i.c.v. [D-Ala2]deltorphin II; following this pretreatment, the [D-Ala2]deltorphin II dose-response line was displaced to the right by more than 37-fold. In contrast, in [D-Ala2]deltorphin II-pretreated mice, the dose-response lines for DPDPE and DAMGO were not altered compared to those obtained in naive animals. Finally, pretreatment with i.c.v. DAMGO produced a rightward displacement of the DAMGO dose-response line of 47-fold, indicating the development of antinociceptive tolerance. In DAMGO-pretreated mice, however, the dose-response lines for DPDPE and [D-Ala2]deltorphin II were not altered compared to those obtained in naive mice. Thus, the data indicate that antinociceptive tolerance develops to DPDPE, [D-Ala2]deltorphin II and DAMGO but that there is no cross-tolerance between these compounds.(ABSTRACT TRUNCATED AT 250 WORDS)
Co-administration of delta opioid agonists at doses which do not produce measurable antinociception were demonstrated to produce an increase in the antinociceptive potency of morphine in the mouse tail-flick test. In contrast, co-administration of equi-antinociceptive combinations of a delta agonist plus morphine for three days resulted in the development of less tolerance to morphine antinociceptive actions. The data indicate that while acute antinociceptive effects of opioid mu agonists are modulated by delta agonists, the development of antinociceptive tolerance is not.
The present study has characterized the antinociceptive actions of [D-Ala2]deltorphin II following intracerebroventricular (i.c.v.) administration in the mouse tail-flick test. [D-Ala2]deltorphin II produced dose- and time-related antinociception, with maximal effects at +10 min and significant antinociception which lasted for 40-60 min. [D-Ala2]deltorphin II was 13-fold more potent than i.c.v. [D-Pen2, D-Pen5]enkephalin (DPDPE), a second highly selective delta agonist, and approximately equipotent with i.c.v. morphine in producing antinociception. The antinociceptive effects of i.c.v. [D-Ala2]deltorphin II and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, ICI 174,864. In contrast, pretreatment with the non-equilibrium mu antagonist, beta-funaltrexamine blocked morphine antinociception, but failed to antagonize [D-Ala2]deltorphin II and DPDPE antinociception. These data indicate that [D-Ala2]deltorphin II produced its antinociceptive effects at a supraspinal delta receptor. [D-Ala2]deltorphin II appears to be the most appropriate delta opioid agonist currently available for studies in vivo and support the involvement of delta receptors in supraspinal antinociception.